Differential efficacy of tyrosine kinase inhibitors according to the types of EGFR mutations and agents in non-small cell lung cancer: a real-world study

Background Both first and second-generation EGFR-TKIs are recommended in advanced NSCLC with common EGFR mutations. However, there are few data on the difference in efficacy of EGFR-TKIs based on the type of EGFR mutation and agents. Methods This retrospective real-world study evaluated the outcomes and clinicopathologic characteristics, including the type of EGFR mutations, of 237 advanced NSCLC patients treated with first- or second-generation (afatinib) EGFR-TKIs as first-line therapy. Results The median progression-free survival (PFS) and overall survival (OS) of all patients were 11 months (M) and 25M, respectively. In the univariate analysis, patients with exon 19 deletion (del) (n=130) had significantly longer median OS compared to those with other mutations (L858R: 84, others: 23) (30 vs. 22 M, p=0.047), without a difference in PFS (p=0.138). Patients treated with afatinib (n=60) showed significantly longer median OS compared to those treated with first-generation TKIs (gefitinib: 159, erlotinib: 18) (30 vs. 23 M, p=0.037), without a difference in PFS (p=0.179). In patients with exon 19 del, there was no significant difference in median PFS (p=0.868) or OS (p=0.361) between patients treated with afatinib and those treated with first-generation TKIs, while significantly better PFS (p=0.042) and trend in OS (p=0.069) were observed in patients receiving afatinib in other mutations. Exon 19 del was independently associated with favorable OS (p=0.028), while age >70 years (p=0.017), ECOG performance status ≥2 (p=0.001), primary metastatic disease (p=0.007), and synchronous brain metastasis (p=0.026) were independent prognostic factors of poor OS. Conclusions The EGFR exon 19 del was associated with favorable OS in advanced NSCLC patients receiving first-line EGFR-TKIs. Moreover, in patients with exon 19 del, first-generation TKIs seem to be a reasonable treatment option if osimertinib is unavailable.


Background
Non-small cell lung cancer (NSCLC) is one of the most common neoplasms, and the leading cause of cancerrelated mortality in several countries, including South Korea [1][2][3].Approximately 65% of patients with NSCLC are diagnosed with advanced status [3], and the clinical outcome of advanced NSCLC with a median overall survival (OS) of 11-22 months remains unsatisfactory, despite advances of palliative chemotherapy [2,4].
Therefore, in the present study, the clinical outcomes of EGFR mutation-positive advanced NSCLC patients treated with first-and second-generation EGFR-TKIs as their first-line treatment were investigated in terms of the EGFR mutation subtypes as well as the agents.

Study population
All EGFR mutation-positive advanced NSCLC patients who started first-line first-or second-generation EGFR-TKIs therapy between July 2011 and June 2018 at our institution were retrospectively identified.The eligibility criteria were cytologically or histologically confirmed NSCLC, and either stage IV based on the 7th edition of the American Joint Committee on Cancer (AJCC) [37] or stage IIIB/recurrent disease unsuitable for definitive local treatment.Some patients and methods of this study cohort were included in previous retrospective studies on EGFR-TKIs in NSCLC [21,38].Nonetheless the criteria for eligibility criteria of this study were slightly different from those of the previous studies, with longer follow-up duration of patients.

Clinical review
The clinical information of eligible patients was retrospectively reviewed.Data collected on the patients included patient characteristics (age, gender, smoking history), performance status (PS) based on the Eastern Cooperative Oncology Group (ECOG) performance scale, histology, disease status at the start of EGFR-TKIs, presence of synchronous brain metastasis, second-or further-line of therapy, and information of survival status.

EGFR mutation analysis
A direct sequencing method was applied for detecting EGFR mutation without routine tumor enrichment.Retrieved Formalin-fixed, paraffin embedded (FFPE) tumor samples were used for genomic DNA extraction by the QIAmp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany).Polymerase chain reaction (PCR) amplification of EGFR exons 18 to 21, using intron-based primers was followed.Sequencing was performed in both the forward and reverse directions.Since September 2014, the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp method has been applied in almost all cases.Genomic DNA of EGFR mutation hot-spots were amplified by PCR with a PNA clamp primer synthesized from a PNA with a wild-type sequence and detected by a fluorescent primer that incorporates locked nucleic acids.This method for preferential amplification of the mutant sequence can detect EGFR mutation in specimens containing 100 to 1000 excess copies of wild-type EGFR sequence [39].

Statistical analysis
The Kaplan-Meier method was used for the calculation of OS and PFS.The time from the start day of the EGFR-TKI treatment to death and the time to disease progression or death by any cause were defined as OS and PFS, respectively.In case of surviving patients at the time of data cut-off with uncertain disease status, the data were censored on the last evaluation date at our institution for PFS.Data on the survivors were censored at the last follow-up for OS.The log-rank test was used for the analysis of the differences between the survival curves.Fisher's exact test was applied to compare categorical variables among the different groups.The joint effects of several variables on survival were determined by the Cox proportional-hazards regression model, including factors with p-values < 0.1 in the univariate analysis.All statistical analyses were performed two-sided using SPSS for Windows 20.0 software.

Statement of ethics
This research protocol was approved by the Institutional Review Board (IRB) of Ajou University Hospital, Suwon, Republic of Korea (AJOUIRB-MDB-2019-394) and all methods were performed in accordance with the relevant guidelines and regulations.The study was designed retrospectively.Written informed consent from patients was not required in accordance with guidelines of the IRB of Ajou University Hospital.

Progression-free and overall survival
The median follow-up duration was 43 (35-103) months for the survivors (42 patients) at the time of analysis.Only one patient was lost to follow-up for survival status after receiving a 14-day prescription of gefitinib and was excluded from the analysis for PFS and OS.The median PFS and OS from the start of EGFR-TKI treatment for all patients were 11 and 25 months, respectively, while those for the 214 patients with EGFR-activating mutation were 12 and 26 months.Patients with exon 19 deletion had significantly longer median OS compared to those with other mutations (30 vs. 22 months, p=0.047,Fig. 1B), without a difference in PFS (12 vs. 9 months, p=0.138,Fig. 1A).Patients treated with afatinib showed significantly longer median OS (30 vs. 23 months, p=0.037,Fig. 2B) compared to those treated with first-generation TKIs, without a difference in PFS (14 vs. 10 months, p=0.179,Fig. 2A).In the multivariate analysis, EGFR exon 19 deletion showed independent association with favorable OS (p=0.028), while age >70 years (p=0.017),ECOG performance status ≥2 (p=0.001),primary metastatic disease (p=0.007), and synchronous brain metastasis (p=0.026) were independent prognostic factors for unfavorable OS (Table 2).
Of the patients who experienced disease progression after first-line EGFR-TKI treatment, those treated with third-generation TKIs demonstrated significantly longer median OS (44 months) from the start of firstline treatment compared to others (183 patients, 20 months, p<0.0001) as well as those who received cytotoxic agents with or without first-or second-generation TKIs (96 patients, 24 months, p=0.006) (Fig. 3).

Discussion
In several retrospective/prospective studies and metaanalyses of those studies, first-generation EGFR-TKI therapy has revealed more favorable outcomes in patients with EGFR exon 19 deletion when compared to those with L858R mutation, especially in terms of PFS [21,24,26,28].Regarding second-generation EGFR-TKIs, an OS survival benefit of afatinib treatment was observed in patients with exon 19 deletion but not those with L858R mutation in a combined analysis of phase III studies (LUX-Lung 3, LUX-Lung 6) comparing cisplatin doublet chemotherapy with afatinib as a first-line setting [40].A few molecular mechanisms were suggested, including higher drug-binding affinity [41,42], different downstream signaling after drug treatment, and lesser baseline  combined T790M mutation in exon 19 deletion [28], for the better efficacy of EGFR-TKIs in exon 19 deletion compared with L858R mutation [41][42][43].However, subsequent retrospective studies and a metanalysis including afatinib and dacomitinib showed no significant differences in outcomes, especially OS, based on the type of EGFR mutation in first-line setting [27,29,36].Similarly, in our cohort, patients with EGFR exon 19 deletion did not show significantly better outcomes compared with those with other mutations when treated with afatinib, while EGFR exon 19 deletion showed longer PFS and OS in patients treated with first-generation EGFR-TKIs, with an independently favorable prognostic significance of exon 19 deletion in terms of OS for all patients.Although the difference in PFS and OS between patients with EGFR exon 19 deletion and other mutations treated with first-generation TKIs is rather small compared with that reported in a previous study on patients with first-line gefitinib treatment, probably due to the longer follow-up duration, which resulted in progression in the majority of patients, the trend toward favorable clinical outcomes in patients with EGFR exon 19 deletion has been observed consistently [21].The lack of improved PFS in patients with exon 19 deletion in the entire cohort may be explained by no significant difference in PFS based on mutation type in the afatinib group.Moreover, the significantly higher proportion of patients treated with thirdgeneration TKIs after progression in exon 19 deletion compared with other mutations may be one of the possible explanations for favorable OS in patients with exon 19 deletion [44].However, this result should be validated in further trials including larger numbers of patients.Furthermore, in our study, patients with EGFR exon 19 deletion revealed almost similar median PFS and OS when they received either first-generation EGFR-TKIs or afatinib, while significantly longer median PFS and a better OS trend were observed in patients with other EGFR mutations receiving afatinib.It remains unclear whether the clinical efficacy of second-generation TKIs is superior to that of first-generation TKIs, as only dacomitinib has demonstrated an OS benefit compared to gefitinib [20].Moreover, with second-generation TKIs, even with dose modification, the incidence of overall and grade ≥ 3 adverse events resulting in negative effects on patients' quality of life (e.g., skin toxicity and diarrhea) is usually higher than that with first-generation TKIs [16][17][18]45].These concerns about the toxicity of afatinib may be reflected in the higher proportion of younger patients and the better performance status of patients treated with afatinib in the present study cohort.Because a proper agent must be selected based on the risk-benefit balance for each patient in clinical practice, the results of present study suggest that first-generation TKIs can be used more safely in poor performance status or elderly patients without compromising clinical efficacy compared to second-generation TKIs, especially those with exon 19 deletion.
In the FLAURA trial, osimertinib resulted in significant prolongation of PFS with a marginal OS benefit compared to first-generation EGFR-TKIs [10,11].However, in Asian patients and those with EGFR L858R mutation, OS benefit of osimertinib was not observed [11].In the present study, patients treated with third-generation TKIs after first-or second-generation TKI failure showed a median OS of 44 months from the start of first-line therapy, comparable to that of osimertinib (38.6 months) in the FLAURA trial [11].Considering that prospective data directly comparing second-and third-generation EGFR-TKIs are not currently available, the results of a few studies including ours suggest that first-line secondgeneration TKIs and sequential third-generation EGFR-TKI treatment may be an effective therapeutic strategy, especially in patients with EGFR L868R mutation [20,46].Overall, first-line first-or second-generation EGFR-TKIs may still be a reasonable choice in routine practice due to its cost-effectiveness [47] in countries where firstline osimertinib is not reimbursable, such as Korea.
The current study demonstrated that EGFR exon 19 deletion was associated independently with favorable OS in advanced NSCLC patients treated with firstline EGFR-TKIs.To the best of our knowledge, current study is the first one showing a significantly favorable OS in patients with EGFR exon 19 deletion, when compared with other mutations in advanced NSCLC patients treated with either first-or second-generation EGFR-TKIs as first-line therapy.Moreover, as this study analyzed every EGFR mutation-positive patient who received first-line first-or second-generation EGFR-TKIs therapy during the defined period with a fairly long follow-up duration (minimum follow-up duration of survivors: 35 months), it reflected the patient outcomes of everyday clinical practice.
However, several limitations were included in this study.First, it was retrospective and performed at a single institution.Second, the number of patients who received third-generation TKIs as second-or further-line therapy was small, as second-or further-line osimertinib treatment has been reimbursable by the Korean national health insurance system since late 2017.Finally, the collection of treatment-related adverse events was not planned considering the retrospective nature of this study.
Nonetheless, several clinical implications can be suggested by the results of our study.First, first-generation EGFR-TKIs could still be recommended as a first-line palliative treatment for NSCLC with EGFR exon 19 deletion, especially in elderly and fragile patients.Second, patients and their families could receive more precise explanations regarding the outcomes and further treatment options after EGFR-TKI therapy based on the types of EGFR mutation.Finally, this study recommends that further clinical trials with EGFR-TKIs should still consider the types of EGFR mutation as a stratification factor.

Conclusions
The EGFR exon 19 deletion was correlated with favorable OS in advanced NSCLC treated with first-line EGFR-TKIs.Moreover, in patients with exon 19 deletion, first-generation TKIs seem to be a reasonable treatment option if osimertinib is unavailable.

Fig. 2 A
Fig. 2 A Progression-free survival and (B) overall survival from the start of EGFR-TKI according to types of tyrosine kinase inhibitors.Exon 19 deletion: 130 patients, others: 106 patients (L858R: 83, uncommon mutations: 18, dual mutations: 5).Censoring was indicated by vertical lines

Table 1
Patient characteristics The baseline characteristics were not statistically different based on the EGFR mutation subtype.However, the proportion of patients who received third-generation TKIs after progression was significantly higher in patients with exon 19 deletion compared to those with other mutations (L858R and uncommon or dual mutations) (Table1).
ECOG Eastern Cooperative Oncology Group, PS Performance status, TKI Tyrosine kinase inhibitor, PD Progressive disease, M Months (median), p p-value *Fisher's exact test a Excluding 17 patients without documentation of PD

Table 2
Univariate and multivariate analysis of progression-free and overall survival PFS Progression-free survival, OS Median overall survival, HR hazard ratio, CI Confidence interval, ECOG Eastern Cooperative Oncology Group, PS Performance status, TKI Tyrosine kinase inhibitor, EGFR Epidermal growth factor receptor; M Months (median), p p-value * Log-rank test †Cox proportional-hazards regression model p=0.045) compared to other mutations, while there was no difference in median PFS and OS based on EGFR mutation subtypes in those treated with afatinib (Table

Table 3
Outcomes according to types of EGFR mutation and tyrosine kinase inhibitorsEGFR Epidermal growth factor receptor, TKI Tyrosine kinase inhibitor, M months (median), p p-value, OS Overall survival, PFS Progression-free survival * Log-rank test